In scientific research, a material can often be characterized by the response of a fluorescent probe to radiation. In some procedures, a sample is illuminated alternately with light of different wavelengths and the fluorescence of the sample with the different illuminating wavelengths is noted. For example, the calcium ion is believed to control a variety of cellular processes with a high degree of spatial and temporal precision. Calcium has been measured in single living cells with high spatial resolution utilizing a microscope and a highly fluorescent calcium sensitive dye Fura-2. A sample to which the dye has been added is illuminated alternately with light of 340 and 380 nanometers. The free fluorescent dye fluoresces at about 500 nanometers maximally in response to the 380 nanometer excitation; whereas, the dye associated with the calcium ion fluoresces at about 500 nanometers maximally in response to the 340 nanometer excitation. The concentration of calcium can then be calculated from the formula: EQU [Ca.sup.++ ].sub.i =K.sub.d [(R-R.sub.min)/(R.sub.max -R)].beta.
Where K.sub.d is the effective dissociation constant for the Fura-2-Calcium reaction, R is the ratio of fluorescent intensity at 500 nm with the 340 and 380 nm excitation, R.sub.min is the limiting value of R at a calcium concentration of zero, R.sub.max is R with fully saturated calcium and .beta. is an optical constant for the system which is a measure of the relative quantum yield at 380 nm of the calcium free and calcium saturated dye.
Often, the distribution of the fluorescent probe or the ratio of the distribution of the probe in its free form relative to its distribution in a bound form within a sample is of interest. For example, the concentration of calcium ions is found to be greater in the cell nucleus than in the cytoplasm. The locations of the ion concentrations have been determined by taking successive two-dimensional images of a sample through incremental focal planes.
The orientation as well as the location of microscopic material may also be significant. For example, alpha-actinin has been observed in muscle cells using fluorescently labeled antibodies specific to alpha-actinin. The location and orientation of the oblong-shaped bodies can be determined by observing the images from plural sections of a sample.